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Naproxen degradation test to monitor Trametes versicolor activity in solid-state bioremediation processes
Journal of Hazardous Materials 179 (2010) 1152–1155
Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Short communication
Naproxen degradation test to monitor Trametes versicolor activity in solid-state bioremediation processes Carlos E. Rodríguez-Rodríguez a,c,∗ , Ernest Marco-Urrea b,1 , Gloria Caminal a a
Unitat de Biocatàlisi Aplicada associada al IQAC (CSIC-UAB), Escola d’Enginyeria, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain Departament d’Enginyeria Química and Institut de Ciència i Tecnologia Ambiental, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain c Centro de Investigación en Contaminación Ambiental, Universidad de Costa Rica, 2060 San José, Costa Rica b
a r t i c l e
i n f o
Article history: Received 29 November 2009 Received in revised form 23 February 2010 Accepted 28 February 2010 Available online 4 March 2010 Keywords: Trametes versicolor Biodegradation test Bioremediation Naproxen Sewage sludge
a b s t r a c t The white-rot fungus Trametes versicolor has been studied as a potential agent for the removal of environmental pollutants. For long-time solid-phase bioremediation systems a test is required to monitor the metabolic status of T. versicolor and its degradation capability at different stages. A biodegradation test based on the percentage of degradation of a spiked model pharmaceutical (anti-inflammatory naproxen) in 24 h (ND24) is proposed to monitor the removal of pharmaceuticals and personal care products in sewage sludge. ND24 is intended to act as a test complementary to ergosterol quantification as specific fungal biomarker, and laccase activity as extracellular oxidative capacity of T. versicolor. For samples collected over 45 d, ND24 values did not necessarily correlate with ergosterol or laccase amounts but in most cases, they were over 30% degradation, indicating that T. versicolor may be suitable for bioremediation of sewage sludge in the studied period. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Trametes versicolor is a white-rot fungus (WRF) capable to biodegrade a wide range of organic pollutants due to its capacity to attack substrates through the action of nonspecific intracellular (i.e. cytochrome P450 system) and extracellular enzymes (i.e. laccases and peroxidases). Given its degrading versatility, the potential use of T. versicolor has been recently studied for the removal of pharmaceuticals and personal care products (PPCP), a group of xenobiotics widespread distributed in the environment at very low concentrations [1,2]. A more recent demonstration of the ability of T. versicolor to colonize sewage sludge in both solid-phase and bioslurry systems and the subsequent degradation of spiked pharmaceuticals in such matrixes open an optimistic horizon for a possible real scale application [3]. Monitoring solid-state bioremediation processes is a very complex task. Time-demanding extraction-based methodologies are required to analyze activity indicators throughout the process, which, in addition, usually provide only indirect proofs of biologi-
∗ Corresponding author at: Unitat de Biocatàlisi Aplicada associada al IQAC (CSICUAB), Escola d’Enginyeria, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. Tel.: +34 93 581 4796; fax: +34 93 581 2013. E-mail address: [email protected] (C.E. Rodríguez-Rodríguez). 1 Current address: Helmholtz Centre for Environmental Research – UFZ, Permoserstrasse, 15, 04318 Leipzig, Germany. 0304-3894/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2010.02.091
cal activity that do not coincide with the removal of pollutants in every case. Therefore the development of tests which allow a more complete knowledge of the physiological status of the fungus in such systems is of great interest. The present research was aimed at defining an activity test to be employed as a complement to laccase activity and ergosterol determinations, to provide wider information on the metabolic status of T. versicolor at different stages through long incubation periods in sewage sludge for PPCP bioremediation purposes. Determination of ergosterol, a specific component of fungal cell membranes, can be a reliable indicator of fungal growth in solid matrixes, however it does not necessarily correlate enzymatic activity or degradation patterns. On the other hand, laccase, an extracellular enzyme produced constitutively in T. versicolor, has been directly associated with the oxidative potential of WRF cultures. Moreover, laccase has been involved in the conversion of several PPCP such as natural and synthetic hormones and antiinflammatory drugs [2,4] and therefore its activity level can provide an indication of the potential transformation of some PPCP in sewage sludge. However, extracellular enzymes may become stabilized on clay or humic substances within the sludge, thus resulting in loss of some activity or extended viability even when the fungal cells have decayed [5]. The test reported here partially overcomes the limitations of the aforementioned indicators by yielding complementary information. It is based on the addition of an external pharmaceutical in a sample and the estimation of its removal after 24 h, which
C.E. Rodríguez-Rodríguez et al. / Journal of Hazardous Materials 179 (2010) 1152–1155
can determine the specific degradation activity of T. versicolor in the sewage sludge. In this case, the pharmaceutical selected was naproxen, an anti-inflammatory drug being extensively used, whose degradation can be catalyzed by laccase and cytochrome P450 in T. versicolor [6]. The naproxen degradation test was applied together with ergosterol and laccase determinations to monitor the activity of T. versicolor in sewage sludge solid-phase cultures. 2. Materials and methods 2.1. Chemicals and fungal strain Naproxen ((S)-(+)-methoxy-␣-methyl-2-naphthalene acetic acid, 98%) and ergosterol (ergosta-5,7,22-trien-3-ol, >95%) were obtained from Sigma–Aldrich Co. (St. Louis, MO). The strain T. versicolor ATCC 42530 was acquired from the American Type Culture Collection, and maintained by subculturing every 30 d on 2% malt extract agar slants (pH 4.5) at 23 ◦ C. T. versicolor blended mycelial suspension was prepared according to MarcoUrrea et al. [7]. 2.2. Sewage sludge and bulking material Dry sewage sludge (17.7% humidity; water holding capacity 0.47 g g−1 dry weight, DW) was obtained from the wastewater treatment plant of El Prat de Llobregat. The plant is located near Barcelona, Spain and it has a total treatment capacity of two million equivalent inhabitants. It is a typical biological activated sludge plant with anaerobic digestion and thermal dehydration. The wheat-straw pellets (ATEA Praha s.r.o., Czech Republic) used as bulking material in solid-phase cultures were kindly provided ˇ Novotny´ and V. Saˇ ˇ sek from the Academy of Science of the by C. Czech Republic. 2.3. Solid-phase cultures Solid-phase cultures with a total dry solid weight content of 2.6 g were performed in 40 mL screw cap vials (28 mm × 95 mm, Grace, Deerfield, IL), containing sewage sludge and 38% bulking material (w/w, dry basis). Wheat-straw pellets were previously hydrated in a 1:2 ratio (material weight:water volume). Each culture was inoculated with 0.25 mL blended mycelium suspension per gram of solid dry weight. Solid content of the cultures was sterilized before mycelium addition. All the cultures were incubated at 25 ◦ C, periodically homogenized and often sprinkled with sterile distilled water to provide moisture. A total of 54 cultures were prepared in parallel to act as unitary samples throughout the experiments. 2.4. Degradation studies Naproxen degradation was evaluated for cultures of different age (10, 17, 25, 31, 38, 45 d). At each time point, triplicate cultures were spiked with a naproxen stock solution to give a final concentration of ∼0.096 mg g DW−1 . Additional triplicate cultures previously autoclaved were employed as heat-killed controls at every time point. After 24 h, the complete content of the cultures was lyophilized (Virtis Sentry freeze-drying equipment, Gardiner, NY) and then subjected to pressurized liquid extraction (PLE) in a PSE-One extractor (Applied Separations, Allentown, PA), as previously described [8]. The volume of extraction was adjusted to 50 mL; a 1.5 mL aliquot was centrifuged at 10,000 rpm for 5 min and the supernatant transferred to amber HPLC vials for subsequent analysis. Quantitative extraction of naproxen was previously verified by this methodology (>95%) [3]. Results were expressed as the percentage of naproxen degradation in 24 h by comparing
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naproxen concentration in the cultures with those in the heat-killed controls. Similarly, the time-course degradation within a period of 24 h was determined for 10 d-old cultures. In parallel, triplicate flasks were sacrificed to analyze ergosterol and laccase activity. 2.5. Analytical methods Ergosterol was measured in homogeneously-mixed samples of solid-phase cultures and extraction was performed as previously described [3]. Analysis was carried out in a Dionex 3000 Ultimate HPLC (Sunnyvale, CA) equipped with an UV detector at 282 nm, using a reverse phase Grace Smart RP18 column (250 mm × 4 mm, particle size 5 m, Deerfield, IL). Methanol was isocratically supplied at 1 mL min−1 as eluent and retention time was ∼6.8 min. Ergosterol content was expressed as milligrams per gram of solid dry weight of culture material (mg g DW−1 ). Laccase was first extracted according to a modified method by Lang et al. [9]: 30 mL sodium acetate buffer (0.16 M, pH 5) were added to 3 g of homogenized sample and shaken for 30 min at 4 ◦ C; extracts of 1.5 mL were centrifuged at 15,000 × g for 15 min and the supernatant was then analyzed. Enzymatic activity was measured using a modified version of the method for manganese peroxidase determination [10]: the reaction mixture consisted of 200 L sodium malonate (250 mM, pH 4.5), 50 L 2,6-dimethoxyphenol (DMP, 20 mM) and 600 L sample. DMP is oxidized by laccase even in the absence of a cofactor. Changes in the absorbance at 468 nm were monitored for 2 min at 30 ◦ C. Results were expressed as activity units (AU) per gram of solid dry weight. One AU was defined as the number of micromoles of DMP oxidized per min. The DMP extinction coefficient was 24,800 M−1 cm−1 . Naproxen analyses were performed using a Dionex 3000 Ultimate HPLC equipped with a UV detector at 230 nm. Chromatographic separation was achieved by injection of 20 L samples on a Grace Smart RP18 column (250 mm × 4 mm, particle size 5 m) and a mobile phase of 65% 6.9 mmol L−1 acetic acid (pH 4.0) plus 35% acetonitrile, added isocratically at 1 mL min−1 [11]. Retention time was ∼14.1 min. Dry weight of solid-phase culture material was determined by drying samples to constant weight in a 100 ◦ C incubator. 3. Results and discussion Studies with T. versicolor have demonstrated promising results for the removal of emerging pollutants in sewage sludge. These potential treatments are thought to be applied for long periods of 30 d or more, given the wide spectrum of contaminants with different degrees of biodegradability present in the sludge. Therefore, the degradation ability of the fungus needs to be assured throughout the whole treatment period. Thus, and based on previous demonstration of depletion mediated by several enzymatic complexes [6], we decided to employ naproxen degradation as a parameter to evaluate metabolic fungal activity during colonization of sewage sludge. Naproxen is a non-prescription analgesic of extensive use, being considered as an emerging pollutant. In the first experiment with 10 d-old culture samples, 31% degradation of spiked naproxen was accomplished within 24 h in sewage sludge solid-phase systems (Fig. 1). Previous studies by our group showed that complete depletion of similar naproxen amounts occurs after 72 h of treatment in 17 d cultures in the same matrix [3], thus indicating that 24 h is an appropriate treatment time for the degradation test, since it assures that not all the pharmaceutical has been removed. This allows, therefore, the comparison of fungal activity in time-course bioremediation experiments. Usually, a test of enzymatic activity determines initial reaction rates which are directly correlated with enzyme concentration. However, in this case degradation may be due to more than one
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C.E. Rodríguez-Rodríguez et al. / Journal of Hazardous Materials 179 (2010) 1152–1155
Fig. 1. Naproxen degradation profiles by T. versicolor in 10 d-old sewage sludge solid-phase cultures containing 38% of bulking material. Treatments: (䊉) heat-killed controls and () live cultures. Values plotted are means ± standard deviations for triplicate cultures.
enzyme and initial degradation rate may be masked by adsorption/diffusion processes mediated by the presence of the fungus. Therefore, and taking advantage of the aforementioned results, the degradation ability of T. versicolor was used to perform the degradation test to monitor the global metabolic status of the fungus. The test was defined as the percentage of naproxen degradation in 24 h under the stated conditions, and was called ND24. To evaluate T. versicolor activity in sewage sludge solid-phase cultures two additional indicators were employed: ergosterol quantification as a component of viable mycelium [12,13] and laccase activity as constitutive enzyme involved in degradation machinery [4]. Results showing the follow up of ergosterol, laccase and the ND24 test are presented in Fig. 2. As it can be seen, important naproxen degradation was achieved along the whole experiment. A minimum value of ND24 appeared at 25 d, time which corresponds to the end of an ergosterol-decrease period. Moreover, a posterior ergosterol increase correlated with a new ND24 increase. It must be pointed out that similar tendencies have been observed in ergosterol content profiles [3]. Important ergosterol amounts were observed during 45 d, indicating the presence of physiologically active T. versicolor biomass. Laccase activity showed a maximum peak and a posterior slowly decay, though notable activity levels were maintained during all the experiment. Determination of laccase is an indicator of extracellular oxidative capacity in T. versicolor. However this activity may last even after metabolic inactivation of the fungus. Contrary, laccase may be produced but inactivated by environment conditions such as high pH [14], making it undetectable in activity tests even if the fungus is still active. In both cases correlation between enzyme and degradation ability may not be achieved. On the other hand, ergostrol quantification, besides being highly laborious, gives information about viability of the fungus; however production of enzymes may depend on nutritional conditions of the substrate or culture medium. The test proposed measures in a global way the degrading-metabolic activity of T. versicolor. Although ND24 does not evaluate the whole range of metabolic pathways, but the ones implicated in the degradation of the selected pollutant, its objective is minimizing the limitations listed above and being complementary to laccase and ergosterol determinations. Results of ND24 showed that all the cultures tested were capable to degrade naproxen at an important extent, over 31% (except the 25 d culture), with a maximum degradation of 56% in the 17 d
Fig. 2. Evolution of ND24 (A), laccase activity (B) and ergosterol content (C) by T. versicolor in solid-phase cultures of different age. Values plotted are means ± standard deviations for triplicate cultures.
cultures, thus demonstrating biodegradation ability in T. versicolor for the whole study period. ND24 indicated that in spite of laccase decay, notable degradation can be accomplished. Fig. 3 presents the profiles of laccase and ergosterol in terms of ND24 values. As we mentioned previously, naproxen is known to be degraded by the intracellular enzyme cytochrome P450 and laccase and therefore a highest correlation between the increase of ND24 and the increase of both laccase and ergosterol content would be expected. However, factors such as the bioavailability of naproxen due to sorption into sludge, for example, could explain low ND24 values at high ergosterol contents or the fact that similar laccase activities resulted in different degradation values. Additionally, it should be taken into account that relatively high variations within the triplicates were obtained in some cases (Fig. 2), which resulted in
C.E. Rodríguez-Rodríguez et al. / Journal of Hazardous Materials 179 (2010) 1152–1155
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Acknowledgments This work was supported by MMAMRM (project 010/PC08/304). The Department of Chemical Engineering of the Universitat Autònoma de Barcelona is the Unit of Biochemical Engineering of the Centre de Referència en Biotecnologia de la Generalitat de Catalunya. The authors are members of a Consolidated Research Group of Catalonia (2009-SGR-656). C. RodríguezRodríguez received a predoctoral fellowship from Universidad de Costa Rica and Consejo Superior de Investigaciones Científicas (UCR-CSIC collaboration). References
Fig. 3. Variation of usual indicators of WRF activity as function of ND24 values in sewage sludge solid-phase cultures of T. versicolor. Laccase activity () and ergosterol (䊉).
difficulty to categorically establish direct correlations between the parameters. Since different but complementary information is obtained from the biodegradation test, ND24 may be a useful assay in the follow up of treatments applied in other solid-state matrixes (i.e. soil), whose control is difficult and usually little information of the process is available. Naproxen was chosen for the ND24 assay based on its fast removal by T. versicolor in solid-state, however this kind of test could be performed with other degradable pollutants. Although the ND24 test is based on the profiles of only one pharmaceutical, the results indicate that T. versicolor is suitable for long-time treatments in sewage sludge bioremediation, since depletion ability remains for periods over 1 month. 4. Conclusions T. versicolor’s ability to deplete spiked naproxen in sewage sludge solid-phase cultures, led to the definition of a complementary biodegradation test. The test corresponds to the percentage of naproxen degradation in 24 h, and it was aimed to be employed as an indicator of fungal activity in solid-phase systems. Application in sewage sludge cultures revealed that ND24 values do not necessarily correlate with ergosterol or laccase amounts. However, notable degradation results for periods over 1 month indicate that T. versicolor may be a suitable agent for bioremediation of sewage sludge and could be able to remove pollutants that require long treatments. ND24 is useful as a complementary test to monitor bioremediation processes in solid-state matrixes.
[1] P. Blánquez, B. Guieysse, Continuous biodegradation of 17-estradiol and 17␣-ethynylestradiol by Trametes versicolor, J. Hazard. Mater. 150 (2008) 459–462. [2] E. Marco-Urrea, M. Pérez-Trujillo, T. Vicent, G. Caminal, Ability of white-rot fungi to remove selected pharmaceuticals and identification of degradation products of ibuprofen by Trametes versicolor, Chemosphere 74 (2009) 765–772. [3] C. Rodríguez-Rodríguez, E. Marco-Urrea, G. Caminal, Degradation of naproxen and carbamazepine in spiked sludge by slurry and solid-phase Trametes versicolor systems, Bioresour. Technol. 101 (2010) 2259–2266. [4] H. Cabana, J.P. Jones, S.N. Agathos, Elimination of endocrine disrupting chemicals using white-rot fungi and their lignin modifying enzymes: a review, Eng. Life Sci. 5 (2007) 429–456. [5] M.D. Wallenstein, M.N. Weintraub, Emerging tools for measuring and modeling the in situ activity of soil extracellular enzymes, Soil Biol. Biochem. 40 (2008) 2098–2106. [6] E. Marco-Urrea, M. Pérez-Trujillo, P. Blánquez, T. Vicent, G. Caminal, Biodegradation of the analgesic naproxen by Trametes versicolor and identification of intermediates using HPLC-DAD-MS and NMR, Bioresour. Technol. 101 (2010) 2159–2166. [7] E. Marco-Urrea, T. Parella, X. Gabarrell, G. Caminal, T. Vicent, C.A. Reddy, Mechanistics of trichloroethylene mineralization by the white-rot fungus Trametes versicolor, Chemosphere 70 (2008) 404–410. ´ A. Jelic, ´ M. Petrovic, ´ D. Barceló, Determination of pharmaceuticals [8] J. Radjenovic, in sewage sludge by pressurized liquid extraction (PLE) coupled to liquid chromatography–tandem mass spectrometry (LC–MS/MS), Anal. Bioanal. Chem. 393 (2009) 1685–1695. [9] E. Lang, F. Nerud, F. Zadrazil, Production of ligninolytic enzymes by Pleurotus sp. and Dichomitus squalens in soil and lignocellulose substrate as influenced by soil microorganisms, FEMS Microbiol. Lett. 167 (1998) 239–244. [10] H. Wariishi, K. Valli, M.H. Gold, Manganese (II) oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium, J. Biol. Chem. 267 (1992) 23688–23695. [11] A. Stafiej, K. Pyrzynska, F. Regan, Determination of anti-inflamatory drugs and estrogens in water by HPLC with UV detection, J. Sep. Sci. 30 (2007) 985–991. [12] M. Barajas-Aceves, M. Hassan, R. Tinoco, R. Vázquez-Duhalt, Effect of pollutants on the ergosterol content as indicator of fungal biomass, J. Microbiol. Methods 5 (2002) 227–236. [13] O. Niemenmaa, S. Galkin, A. Hatakka, Ergosterol contents of some wood-rotting basidiomycete fungi grown in liquid and solid cultures conditions, Int. Biodeterior. Biodegrad. 62 (2008) 125–134. ˜ [14] T. Lu-Chau, F.J. Ruiz-Duenas, S. Camarero, G. Feijoo, M.J. Martínez, J.M. Lema, A.T. Martínez, Effect of pH on the stability of Pleurotus eryngii versatile peroxidase during heterologous production in Emericella nidulans, Bioprocess Biosyst. Eng. 26 (2004) 287–293.
Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Short communication
Naproxen degradation test to monitor Trametes versicolor activity in solid-state bioremediation processes Carlos E. Rodríguez-Rodríguez a,c,∗ , Ernest Marco-Urrea b,1 , Gloria Caminal a a
Unitat de Biocatàlisi Aplicada associada al IQAC (CSIC-UAB), Escola d’Enginyeria, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain Departament d’Enginyeria Química and Institut de Ciència i Tecnologia Ambiental, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain c Centro de Investigación en Contaminación Ambiental, Universidad de Costa Rica, 2060 San José, Costa Rica b
a r t i c l e
i n f o
Article history: Received 29 November 2009 Received in revised form 23 February 2010 Accepted 28 February 2010 Available online 4 March 2010 Keywords: Trametes versicolor Biodegradation test Bioremediation Naproxen Sewage sludge
a b s t r a c t The white-rot fungus Trametes versicolor has been studied as a potential agent for the removal of environmental pollutants. For long-time solid-phase bioremediation systems a test is required to monitor the metabolic status of T. versicolor and its degradation capability at different stages. A biodegradation test based on the percentage of degradation of a spiked model pharmaceutical (anti-inflammatory naproxen) in 24 h (ND24) is proposed to monitor the removal of pharmaceuticals and personal care products in sewage sludge. ND24 is intended to act as a test complementary to ergosterol quantification as specific fungal biomarker, and laccase activity as extracellular oxidative capacity of T. versicolor. For samples collected over 45 d, ND24 values did not necessarily correlate with ergosterol or laccase amounts but in most cases, they were over 30% degradation, indicating that T. versicolor may be suitable for bioremediation of sewage sludge in the studied period. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Trametes versicolor is a white-rot fungus (WRF) capable to biodegrade a wide range of organic pollutants due to its capacity to attack substrates through the action of nonspecific intracellular (i.e. cytochrome P450 system) and extracellular enzymes (i.e. laccases and peroxidases). Given its degrading versatility, the potential use of T. versicolor has been recently studied for the removal of pharmaceuticals and personal care products (PPCP), a group of xenobiotics widespread distributed in the environment at very low concentrations [1,2]. A more recent demonstration of the ability of T. versicolor to colonize sewage sludge in both solid-phase and bioslurry systems and the subsequent degradation of spiked pharmaceuticals in such matrixes open an optimistic horizon for a possible real scale application [3]. Monitoring solid-state bioremediation processes is a very complex task. Time-demanding extraction-based methodologies are required to analyze activity indicators throughout the process, which, in addition, usually provide only indirect proofs of biologi-
∗ Corresponding author at: Unitat de Biocatàlisi Aplicada associada al IQAC (CSICUAB), Escola d’Enginyeria, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. Tel.: +34 93 581 4796; fax: +34 93 581 2013. E-mail address: [email protected] (C.E. Rodríguez-Rodríguez). 1 Current address: Helmholtz Centre for Environmental Research – UFZ, Permoserstrasse, 15, 04318 Leipzig, Germany. 0304-3894/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2010.02.091
cal activity that do not coincide with the removal of pollutants in every case. Therefore the development of tests which allow a more complete knowledge of the physiological status of the fungus in such systems is of great interest. The present research was aimed at defining an activity test to be employed as a complement to laccase activity and ergosterol determinations, to provide wider information on the metabolic status of T. versicolor at different stages through long incubation periods in sewage sludge for PPCP bioremediation purposes. Determination of ergosterol, a specific component of fungal cell membranes, can be a reliable indicator of fungal growth in solid matrixes, however it does not necessarily correlate enzymatic activity or degradation patterns. On the other hand, laccase, an extracellular enzyme produced constitutively in T. versicolor, has been directly associated with the oxidative potential of WRF cultures. Moreover, laccase has been involved in the conversion of several PPCP such as natural and synthetic hormones and antiinflammatory drugs [2,4] and therefore its activity level can provide an indication of the potential transformation of some PPCP in sewage sludge. However, extracellular enzymes may become stabilized on clay or humic substances within the sludge, thus resulting in loss of some activity or extended viability even when the fungal cells have decayed [5]. The test reported here partially overcomes the limitations of the aforementioned indicators by yielding complementary information. It is based on the addition of an external pharmaceutical in a sample and the estimation of its removal after 24 h, which
C.E. Rodríguez-Rodríguez et al. / Journal of Hazardous Materials 179 (2010) 1152–1155
can determine the specific degradation activity of T. versicolor in the sewage sludge. In this case, the pharmaceutical selected was naproxen, an anti-inflammatory drug being extensively used, whose degradation can be catalyzed by laccase and cytochrome P450 in T. versicolor [6]. The naproxen degradation test was applied together with ergosterol and laccase determinations to monitor the activity of T. versicolor in sewage sludge solid-phase cultures. 2. Materials and methods 2.1. Chemicals and fungal strain Naproxen ((S)-(+)-methoxy-␣-methyl-2-naphthalene acetic acid, 98%) and ergosterol (ergosta-5,7,22-trien-3-ol, >95%) were obtained from Sigma–Aldrich Co. (St. Louis, MO). The strain T. versicolor ATCC 42530 was acquired from the American Type Culture Collection, and maintained by subculturing every 30 d on 2% malt extract agar slants (pH 4.5) at 23 ◦ C. T. versicolor blended mycelial suspension was prepared according to MarcoUrrea et al. [7]. 2.2. Sewage sludge and bulking material Dry sewage sludge (17.7% humidity; water holding capacity 0.47 g g−1 dry weight, DW) was obtained from the wastewater treatment plant of El Prat de Llobregat. The plant is located near Barcelona, Spain and it has a total treatment capacity of two million equivalent inhabitants. It is a typical biological activated sludge plant with anaerobic digestion and thermal dehydration. The wheat-straw pellets (ATEA Praha s.r.o., Czech Republic) used as bulking material in solid-phase cultures were kindly provided ˇ Novotny´ and V. Saˇ ˇ sek from the Academy of Science of the by C. Czech Republic. 2.3. Solid-phase cultures Solid-phase cultures with a total dry solid weight content of 2.6 g were performed in 40 mL screw cap vials (28 mm × 95 mm, Grace, Deerfield, IL), containing sewage sludge and 38% bulking material (w/w, dry basis). Wheat-straw pellets were previously hydrated in a 1:2 ratio (material weight:water volume). Each culture was inoculated with 0.25 mL blended mycelium suspension per gram of solid dry weight. Solid content of the cultures was sterilized before mycelium addition. All the cultures were incubated at 25 ◦ C, periodically homogenized and often sprinkled with sterile distilled water to provide moisture. A total of 54 cultures were prepared in parallel to act as unitary samples throughout the experiments. 2.4. Degradation studies Naproxen degradation was evaluated for cultures of different age (10, 17, 25, 31, 38, 45 d). At each time point, triplicate cultures were spiked with a naproxen stock solution to give a final concentration of ∼0.096 mg g DW−1 . Additional triplicate cultures previously autoclaved were employed as heat-killed controls at every time point. After 24 h, the complete content of the cultures was lyophilized (Virtis Sentry freeze-drying equipment, Gardiner, NY) and then subjected to pressurized liquid extraction (PLE) in a PSE-One extractor (Applied Separations, Allentown, PA), as previously described [8]. The volume of extraction was adjusted to 50 mL; a 1.5 mL aliquot was centrifuged at 10,000 rpm for 5 min and the supernatant transferred to amber HPLC vials for subsequent analysis. Quantitative extraction of naproxen was previously verified by this methodology (>95%) [3]. Results were expressed as the percentage of naproxen degradation in 24 h by comparing
1153
naproxen concentration in the cultures with those in the heat-killed controls. Similarly, the time-course degradation within a period of 24 h was determined for 10 d-old cultures. In parallel, triplicate flasks were sacrificed to analyze ergosterol and laccase activity. 2.5. Analytical methods Ergosterol was measured in homogeneously-mixed samples of solid-phase cultures and extraction was performed as previously described [3]. Analysis was carried out in a Dionex 3000 Ultimate HPLC (Sunnyvale, CA) equipped with an UV detector at 282 nm, using a reverse phase Grace Smart RP18 column (250 mm × 4 mm, particle size 5 m, Deerfield, IL). Methanol was isocratically supplied at 1 mL min−1 as eluent and retention time was ∼6.8 min. Ergosterol content was expressed as milligrams per gram of solid dry weight of culture material (mg g DW−1 ). Laccase was first extracted according to a modified method by Lang et al. [9]: 30 mL sodium acetate buffer (0.16 M, pH 5) were added to 3 g of homogenized sample and shaken for 30 min at 4 ◦ C; extracts of 1.5 mL were centrifuged at 15,000 × g for 15 min and the supernatant was then analyzed. Enzymatic activity was measured using a modified version of the method for manganese peroxidase determination [10]: the reaction mixture consisted of 200 L sodium malonate (250 mM, pH 4.5), 50 L 2,6-dimethoxyphenol (DMP, 20 mM) and 600 L sample. DMP is oxidized by laccase even in the absence of a cofactor. Changes in the absorbance at 468 nm were monitored for 2 min at 30 ◦ C. Results were expressed as activity units (AU) per gram of solid dry weight. One AU was defined as the number of micromoles of DMP oxidized per min. The DMP extinction coefficient was 24,800 M−1 cm−1 . Naproxen analyses were performed using a Dionex 3000 Ultimate HPLC equipped with a UV detector at 230 nm. Chromatographic separation was achieved by injection of 20 L samples on a Grace Smart RP18 column (250 mm × 4 mm, particle size 5 m) and a mobile phase of 65% 6.9 mmol L−1 acetic acid (pH 4.0) plus 35% acetonitrile, added isocratically at 1 mL min−1 [11]. Retention time was ∼14.1 min. Dry weight of solid-phase culture material was determined by drying samples to constant weight in a 100 ◦ C incubator. 3. Results and discussion Studies with T. versicolor have demonstrated promising results for the removal of emerging pollutants in sewage sludge. These potential treatments are thought to be applied for long periods of 30 d or more, given the wide spectrum of contaminants with different degrees of biodegradability present in the sludge. Therefore, the degradation ability of the fungus needs to be assured throughout the whole treatment period. Thus, and based on previous demonstration of depletion mediated by several enzymatic complexes [6], we decided to employ naproxen degradation as a parameter to evaluate metabolic fungal activity during colonization of sewage sludge. Naproxen is a non-prescription analgesic of extensive use, being considered as an emerging pollutant. In the first experiment with 10 d-old culture samples, 31% degradation of spiked naproxen was accomplished within 24 h in sewage sludge solid-phase systems (Fig. 1). Previous studies by our group showed that complete depletion of similar naproxen amounts occurs after 72 h of treatment in 17 d cultures in the same matrix [3], thus indicating that 24 h is an appropriate treatment time for the degradation test, since it assures that not all the pharmaceutical has been removed. This allows, therefore, the comparison of fungal activity in time-course bioremediation experiments. Usually, a test of enzymatic activity determines initial reaction rates which are directly correlated with enzyme concentration. However, in this case degradation may be due to more than one
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Fig. 1. Naproxen degradation profiles by T. versicolor in 10 d-old sewage sludge solid-phase cultures containing 38% of bulking material. Treatments: (䊉) heat-killed controls and () live cultures. Values plotted are means ± standard deviations for triplicate cultures.
enzyme and initial degradation rate may be masked by adsorption/diffusion processes mediated by the presence of the fungus. Therefore, and taking advantage of the aforementioned results, the degradation ability of T. versicolor was used to perform the degradation test to monitor the global metabolic status of the fungus. The test was defined as the percentage of naproxen degradation in 24 h under the stated conditions, and was called ND24. To evaluate T. versicolor activity in sewage sludge solid-phase cultures two additional indicators were employed: ergosterol quantification as a component of viable mycelium [12,13] and laccase activity as constitutive enzyme involved in degradation machinery [4]. Results showing the follow up of ergosterol, laccase and the ND24 test are presented in Fig. 2. As it can be seen, important naproxen degradation was achieved along the whole experiment. A minimum value of ND24 appeared at 25 d, time which corresponds to the end of an ergosterol-decrease period. Moreover, a posterior ergosterol increase correlated with a new ND24 increase. It must be pointed out that similar tendencies have been observed in ergosterol content profiles [3]. Important ergosterol amounts were observed during 45 d, indicating the presence of physiologically active T. versicolor biomass. Laccase activity showed a maximum peak and a posterior slowly decay, though notable activity levels were maintained during all the experiment. Determination of laccase is an indicator of extracellular oxidative capacity in T. versicolor. However this activity may last even after metabolic inactivation of the fungus. Contrary, laccase may be produced but inactivated by environment conditions such as high pH [14], making it undetectable in activity tests even if the fungus is still active. In both cases correlation between enzyme and degradation ability may not be achieved. On the other hand, ergostrol quantification, besides being highly laborious, gives information about viability of the fungus; however production of enzymes may depend on nutritional conditions of the substrate or culture medium. The test proposed measures in a global way the degrading-metabolic activity of T. versicolor. Although ND24 does not evaluate the whole range of metabolic pathways, but the ones implicated in the degradation of the selected pollutant, its objective is minimizing the limitations listed above and being complementary to laccase and ergosterol determinations. Results of ND24 showed that all the cultures tested were capable to degrade naproxen at an important extent, over 31% (except the 25 d culture), with a maximum degradation of 56% in the 17 d
Fig. 2. Evolution of ND24 (A), laccase activity (B) and ergosterol content (C) by T. versicolor in solid-phase cultures of different age. Values plotted are means ± standard deviations for triplicate cultures.
cultures, thus demonstrating biodegradation ability in T. versicolor for the whole study period. ND24 indicated that in spite of laccase decay, notable degradation can be accomplished. Fig. 3 presents the profiles of laccase and ergosterol in terms of ND24 values. As we mentioned previously, naproxen is known to be degraded by the intracellular enzyme cytochrome P450 and laccase and therefore a highest correlation between the increase of ND24 and the increase of both laccase and ergosterol content would be expected. However, factors such as the bioavailability of naproxen due to sorption into sludge, for example, could explain low ND24 values at high ergosterol contents or the fact that similar laccase activities resulted in different degradation values. Additionally, it should be taken into account that relatively high variations within the triplicates were obtained in some cases (Fig. 2), which resulted in
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Acknowledgments This work was supported by MMAMRM (project 010/PC08/304). The Department of Chemical Engineering of the Universitat Autònoma de Barcelona is the Unit of Biochemical Engineering of the Centre de Referència en Biotecnologia de la Generalitat de Catalunya. The authors are members of a Consolidated Research Group of Catalonia (2009-SGR-656). C. RodríguezRodríguez received a predoctoral fellowship from Universidad de Costa Rica and Consejo Superior de Investigaciones Científicas (UCR-CSIC collaboration). References
Fig. 3. Variation of usual indicators of WRF activity as function of ND24 values in sewage sludge solid-phase cultures of T. versicolor. Laccase activity () and ergosterol (䊉).
difficulty to categorically establish direct correlations between the parameters. Since different but complementary information is obtained from the biodegradation test, ND24 may be a useful assay in the follow up of treatments applied in other solid-state matrixes (i.e. soil), whose control is difficult and usually little information of the process is available. Naproxen was chosen for the ND24 assay based on its fast removal by T. versicolor in solid-state, however this kind of test could be performed with other degradable pollutants. Although the ND24 test is based on the profiles of only one pharmaceutical, the results indicate that T. versicolor is suitable for long-time treatments in sewage sludge bioremediation, since depletion ability remains for periods over 1 month. 4. Conclusions T. versicolor’s ability to deplete spiked naproxen in sewage sludge solid-phase cultures, led to the definition of a complementary biodegradation test. The test corresponds to the percentage of naproxen degradation in 24 h, and it was aimed to be employed as an indicator of fungal activity in solid-phase systems. Application in sewage sludge cultures revealed that ND24 values do not necessarily correlate with ergosterol or laccase amounts. However, notable degradation results for periods over 1 month indicate that T. versicolor may be a suitable agent for bioremediation of sewage sludge and could be able to remove pollutants that require long treatments. ND24 is useful as a complementary test to monitor bioremediation processes in solid-state matrixes.
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